Carcinostatic compound and production thereof

ABSTRACT

PCT No. PCT/JP92/01017 Sec. 371 Date Feb. 8, 1994 Sec. 102(e) Date Feb. 8, 1994 PCT Filed Aug. 7, 1992 PCT Pub. No. WO93/03039 PCT Pub. Date Feb. 18, 1993Described are stelleramacrin A and stelleramacrin B represented by the following formulas (I) and (II), respectively:    &lt;IMAGE&gt;  (I)  &lt;IMAGE&gt;  (II)  a process for the preparation thereof; and anticancer agents containing either of them as an active ingredient. Therapeutics for solid cancer, said therapeutics containing either of gnidimacrin or pimelea factor P2 as an active ingredient, are also described.

This application is a 371 of PCT/JP92/01017 filed Aug. 7, 1992.

TECHNICAL FIELD

The present invention relates to anticancer compounds and a preparationprocess thereof, and more specifically to novel anticancer compoundscontained in Rui-xiang-lang-du, that is, a Chinese herbal and crudedrug, and a preparation process thereof.

BACKGROUND ART

Chinese herbal and crude drugs can be stated as the fruit oflong-established wisdom of human beings. Confirming the existence ofstrong carcinostatic activities in Rui-xiang-lang-du (Stellerachamaejasme. L, Euphorbia fischeriana Steud., E. ebiacteola Hayata) inthe course of a study on carcinostatic Chinese herbal and crude drugs,the essences of whose carcinostatic activities have not yet beenidentified, the present inventors filed a patent application on aprocess for the collection of its carcinostatic substance (JapanesePatent Application No. 58126/1990). The application, however, did notdeal to such an extent as efficiently isolating a compound havingcarcinostatic activities and confirming its effectiveness against solidcancer.

DISCLOSURE OF THE INVENTION

The present inventors have proceeded with further research on acarcinostatically active component contained in the carcinostaticsubstance of Rui-xiang-lang-du, resulting in successful isolation of acomponent contributing to the carcinostatic activities ofRui-xiang-lang-du. In addition, the component has also been found tocontain a novel compound represented by the formula described below.

Namely, a first object of the present invention is to providestelleramacrin A and stelleramacrin B represented by the followingformulas (I) and (II), respectively: ##STR2##

A second object of the present invention is to provide a process for thepreparation of stelleramacrin A or stelleramacrin B fromRui-xiang-lang-du.

Another object of the present invention is to provide an anticanceragent containing stelleramacrin A or stelleramacrin B.

A further object of the present invention is to provide a therapeuticfor solid cancer, said therapeutic containing gnidimacrin or pimeleafactor P₂ as an active ingredient.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a proton NMR spectrum of stelleramacrin A according to thepresent invention.

FIG. 2 is a proton NMR spectrum of stelleramacrin

BEST MODES FOR CARRYING OUT THE INVENTION

Stelleramacrin A and stelleramacrin B (which may hereinafter becollectively called "stelleramacrin") according to the presentinvention, which are represented by the formulas (I) and (II),respectively, can each be obtained by extracting an aerial orsubterranean part (rootstock) of Rui-xiang-lang-du with an organicsolvent and then purifying the extract.

Described specifically, an aerial or subterranean part ofRui-xiang-lang-du, for example, is extracted with a lower alcohol suchas methanol, ethanol or propanol and the extract is then extracted witha water-insoluble organic solvent such as petroleum ether to obtain acrude extract. In the next place, the crude extract is purified bychromatography on a silica gel column while using hexane-ethyl acetateor the like as an eluent, chromatography on an ODS column while usingmethanol-water, or a like method, whereby some active fractions areobtained. The active fractions are subjected to high-performance liquidchromatography and preparative thin-layer chromatography for furtherpurification, whereby stelleramacrin represented by the formula (I) or(II) can be isolated as a compound having carcinostatic activities.

Although anticancer compounds other than stelleramacrin, for example,gnidimacrin [J. Nat. Prod. 1985, 48(3), 440-445], pemelea factor P₂(Abstracts of Lectures addressed at Natural Organic Compounds Forum, 27,734-741), subtoxin A (Abstracts of Lectures addressed at Natural OrganicCompounds Forum, 27, 734-741), huratoxin (Abstracts of Lecturesaddressed at Natural Organic Compounds Forum, 27, 734-741) and simplexin[Aust. Vet. J., 51(6), 325-326] are also contained, stelleramacrin isobtained in fractions different from those of such other anticancercompounds in the separation and purification steps according to theprocess described above. Its structure is determined by analyzing thesame on the basis of its ultraviolet absorption spectrum, proton nucleusmagnetic resonance spectrum, mass spectrum or the like.

Incidentally, gnidimacrin, pimelea factor P₂ and the like which are alsoanticancer compounds obtained from Rui-xiang-lang-du are knownsubstances. Such known substances have long been regarded effective forthe therapy of ascites-type leukemia. As can be understood easily fromthe therapeutic cases by carbazylquinone and bleomycin, for example, nocorrelation has been recognized between action against leukemia andcarcinostatic activities against solid cancer. It was not known at allabout whether or not these compounds have carcinostatic activitiesagainst solid cancer.

As a result of the study by the present inventors, it has been found forthe first time that gnidimacrin and pimelea factor P₂ also have stronginhibiting action against solid cancer such as gastric cancer, pulmonarycancer, liver cancer or the like.

When stelleramacrin according to the present invention is employed as ananticancer agent or when gnidimacrin or pimelea factor P₂ is used as atherapeutic for solid cancer, each of these compounds can beadministered to animals or men neat or in combination with one or moreconventionally-employed, pharmaceutically-acceptable carriers.

No particular limitation is imposed on the administration form ofstelleramacrin, gnidimacrin and pimelea factor P₂, so that a suitableadministration form can be selected as need. These compounds may each beadministered either as an oral preparation such as tablets, capsules,granules, fine subtilaes, powders or the like, or as a parenteralpreparation such as injections or suppositories.

Stelleramacrin A, a novel compound prepared according to the presentinvention, has an LD₅₀ value of 5 mg/kg while stelleramacrin B, also anovel compound prepared according to the present invention, has an LD₅₀value of 12 mg/kg. When these compounds are used as oral preparations,their dosage varies depending on the age, body weight and conditions ofeach patient. To bring about intended effects, stelleramacrin may beadded. in an amount of about 0.02 mg to 20 mg a day per adult in severalportions.

It is proper, on the other hand, to administer gnidimacrin or pimeleafactor P₂ in an amount of about 0.01 to 10 mg a day per adult.

Oral preparations can be formulated in a manner known per se in the artby using, as an excipient, starch, lactose, sucrose, mannitol,caboxymethylcellulose, corn starch or an inorganic salt. For theformulation of oral preparations, one or more of binders,disintegrators, surfactants, lubricants, fluidity improvers, corrigents,colorants, perfumes and the like can also be added. Specific examples ofthese additives will be illustrated hereinafter.

Examples of the binder include starch, dextrin, gum arabic powder,gelatin, hydroxypropyl starch, methylcellulose, sodiumcarboxymethylcellulose, hydroxypropylcellulose, crystalline cellulose,ethylcellulose, polyvinyl pyrrolidone and macrogol.

Examples of the disintegrator include starch, hydroxypropyl starch,sodium carboxymethylcellulose, calcium carboxymethylcellulose,carboxymethylcellulose and low-substituted hydroxypropylcellulose.

Illustrative surfactants include sodium laurylsulfate, soybean lecithin,sucrose fatty acid esters and polysorbate 80.

Exemplary lubricants include talc, waxes, hydrogenated vegetable oils,sucrose fatty acid esters, magnesium stearate, calcium stearate,aluminum stearate and polyethylene glycol.

Examples of the fluidity improver include light silicic anhydride, driedaluminum hydroxide gel, synthetic aluminum silicate and magnesiumsilicate.

The compounds, which pertain to the present invention, can also beadministered as suspensions, emulsions, syrups or elixirs. Thesepreparation forms may contain a corrigent, colorant and/or the like.

When the compounds according to the present invention are employed asparenteral preparations, their dosage to bring about intended effectsvaries depending on the age, body weight and conditions of the patient.In general, it is necessary to administer about 0.005 mg to 5 mg a day,in terms of the weight of each compound, per adult by intravenousinjection, intravenous drip infusion, subcutaneous injection orintramuscular injection.

These parenteral preparations can be formulated in a manner known per sein the art. Illustrative usable diluents include distilled water forinjection, physiological saline, an aqueous glucose solution, vegetableoil for injection, sesame oil, peanut oil, soybean oil, corn oil,propylene glycol and polyethylene glycol. Sterilizers, antiseptics,stabilizers and/or the like can also be added as needed.

As a preferred method from the viewpoint of stability, it is alsopossible to fill such a parenteral preparation in vials, and then tofreeze it to remove its water, thereby forming it into a lyophilizate bya usual lyophillization technique. Immediately before use, thelyophillizate can be reconstituted into a liquid preparation.

Isotonicities, stabilizers, antiseptics, soothing agents and/or the likecan also be added as needed.

Further examples of the parenteral preparation include coatingpreparations such as external liquid preparations and ointments andsuppositories for rectal administration. They can be formulated in amanner known per se in the art.

The present invention will hereinafter be described in further detail bythe following Examples.

EXAMPLE 1

A subterranean part (1 kg) of Stellera chamejasme. L was pulverized,followed by the addition of 3 1 of methanol. The resultant mixture washeated for 5 hours under. reflux for extraction. The residue of theextraction was extracted again under the same conditions. The bothextracts were combined together and then filtered under reducedpressure, whereby 153.6 g of methanol extract were obtained. Themethanol extract was extracted further with petroleum ether for 5 hoursin a Soxhlet extractor, whereby 15.8 g of a petroleum ether extract(MMP) were obtained.

The resultant MMP (6.5 g) was subjected to chromatography on a silicagel column (5 cm in diameter and 16.5 cm in length), followed by thesuccessive eluation with hexane-ethyl acetate as an eluent [1:4(v/v),1:2(v/v), 1:1(v/v)] to effect fractionation. Eluate fractions (1 g)corresponding to the 1:1 (v/v) mixed solvent of hexane and ethyl acetatewere subjected to high-performance liquid chromatography on an ODSreversed phase column (0.2 cm in diameter, 25 cm in length), followed bythe eluation with a 10:1 (v/v) mixed solvent of methanol and water. As aresult, confirmed was the existence of substances having strongcarcinostatic activities in fractions which were obtained at retentiontimes of 9.5 min, 11.4 min, 14.8 min, 17.2 min, 19.8 min and 21.0 min,respectively. These fractions were designated as Fraction 1, Fraction 2,Fraction 3, Fraction 4, Fraction 5 and Fraction 6 in the increasingorder of the retention times.

Each fraction was thereafter purified by repeating preparativethin-layer chromatography on a silica gel column [hexane:ethylacetate=1:2 (v/v)] and preparative high-performance liquidchromatography on an ODS column [methanol:water=10:1 (v/v)] as manytimes as needed, whereby a single compound was isolated. The yields ofthe single compounds so isolated were 1.90 mg from Fraction 1,3.29 mgfrom Fraction 2, 2.06 mg from Fraction 3, 8.23 mg from Fraction 4 and1.23 mg from Fraction 5. When an aerial part of Rui-xiang-lang-du wasused instead as a raw material, obtained were 1.35 mg from Fraction 1and 3.61 mg from Fraction 2 in exactly the same manner.

As a result of determination of the chemical structure of each of thecompounds so isolated, the compound isolated from Fraction 1 was foundto be a novel compound represented by the formula (1). This compound wasnamed "stelleramacrin A" by the present inventors. The compound isolatedfrom Fraction 2 was identified as gnidimacrin, that from Fraction 3 aspimelea factor P₂, that from Fraction 4 as subtoxin A, that fromFraction 5 as huratoxin and that from Fraction 6 as simplexin.

Physicochemical properties of stelleramacrin, the novel compound, amongthe isolated compounds will be described below:

Physicochemical properties of stelleramacrin:

Molecular formula: C₃₇ H₅₀ O₁₁

Appearance: white powder

Mass analysis: M+H=671

Benzoyl group: 1

Daphnan-system: the skeleton of diterpene is contained.

Proton nuclear magnetic resonance spectrum (δppm in CDCl₃) ¹ H-NMR:2.59(1H,dd,J=12.5), 1.65(1H,m), 3.81(1H,d), 3.72-3.95(1H), 3.39(1H,s),3.03 (1H,d,J=2.9), 2.91(1H,d,J=12.5), 2.76(1H,m), 2.02(1H,d, J=7.5),2.32(1H,d,J=14.7), 4.40(1H, d,J=2.9), 5.19(1H,s), 4.95(1H,s),1.84(3H,s), 4.92(1H,bd,J=10.0), 4.37(1H,t,J=10.0), 1.17 (3H,d,J=7.6),3.89(1H,d,J=10.0), 3.73(1H,d, J=11), 2.26(1H,m), 3.78-3.89(1H,d,J=7.3),1.00(3H,d,J=7.6), 8.07(1H,m), 7.57 (1H,m), 7.47 (2H,m)

Carbon nuclear magnetic resonance spectrum (δppm in CDCl₃) ¹³ C-NMR:14.58, 18.20, 19.05, 22.81, 23.11, 23.61, 24.19, 25.18, 27.25, 28.52,29.51, 36.53, 37.62, 40.85, 48.01, 48.13, 61.28, 63.18, 65.12, 68.27,70.81, 72.08, 78.47, 79.42, 81.03, 81.34, 84.42, 111.89, 118.50, 128.43,129.73, 130.30, 133.13, 145.56, 167.09.

EXAMPLE 2

MMP (2 g), which had been obtained in the manner described in Example 1,was subjected to open-column chromatography (ODS column,methanol:water=10:1, v/v) employing as a carrier ODS (LC sorb,"Sp-A-ODS"; product of KEMCO CORP), followed by the eluation with a 9:1(v/v) mixed eluate of methanol and water. As a result of detection ofthe eluate so obtained at 254 nm, six fractions were obtained. It wasconfirmed that among them, Fraction 1, 2 and 3 had strong carcinostaticactivities- Fraction 2, which showed the strongest carcinostaticactivities, was subjected further to preparative high-performance liquidchromatography on a Shim-Pack pREP-ODS column (20 mm×25 cm), followed bythe eluation with a 10:1 (v/v) mixed solvent of methanol and water whilemonitoring fractions by UV at 228 nm. As a result, stelleramacrin A,gnidimacrin and stelleramacrin B were found to be eluted at eluationtimes of 17.5 min, 27.9 min and 44.4 min, respectively.

Proton NMR spectra of stelleramacrin A and stelleramacrin B so obtainedare shown in FIG. 1 and FIG. 2, respectively.

In addition, stelleramacrin was also obtained from Fraction 1 andFraction 3 described above.

Test 1

The mouse leukemia cell P-388 was intraperitoneally transplanted to BDF₁mice in an amount of 1×10⁶ cells per mouse. From the following day, eachtest drug was intraperitoneally administered to the mice once a day for5 days. Each test group was compared in the number of survival days witha control group to determine an apothanasia rate. The results are shownin Table 1.

                  TABLE 1                                                         ______________________________________                                                   Dosage   Average number of                                                                           Apothanasia                                 Test drug  (mg/kg)  survival days (days)                                                                        rate (%)                                    ______________________________________                                        Control group       8.08                                                      Stelleramacrin A                                                                         1        13.83         71                                          Stelleramacrin B                                                                         1        12.17         48                                          ______________________________________                                    

Test 2

Cells of gastric cancer, pulmonary cancer, liver cancer and leukemia,which had been separated from human patients, were separately placed inportions of RMP-1640 medium containing 10% calf serum and then incubatedat 37° C. for 4 days in a 5% CO₂ -incubator to obtain a cell populationof 10⁴ /ml. To the cells of each cancer kind so cultured, stelleramacrinwas added at varied concentrations, and the concentration (IC₅₀) of eachtest compound at which proliferation of the human cancer cells wasinhibited by 50% was deterimined. The results are shown in Table 2.

                  TABLE 2                                                         ______________________________________                                               Kind of IC.sub.50 (μg/ml)                                           Site of cancer                                                                         cancer    Stelleramacrin A                                                                           Stelleramacrin B                              ______________________________________                                        Gastric  MKU-28    0.2          1                                             cancer   MKV-74    >10          >10                                                    K-III     0.1          1.5                                                    MKV-45    0.8          2.7                                           Pulmonary                                                                              LU-99     >10          >10                                           cancer   N-237     5.3          >10                                                    H-69      10.2         >10                                                    PC-14     8.4          >10                                                    PC-7      5.1          7.3                                           Liver cancer                                                                           HLE       >10          >10                                           Leukemia K-502     10           >10                                           ______________________________________                                    

Test 3

Hypodermically transplanted to induce solid cancer were Lewis pulmonarycancer cells to BDF₁ mice in an amount of 1×10⁶ cells per mouse, Clon 26large bowel cancer cells to BALB/C mice in an amount of 5×10⁵ cells permouse and a 20% homogenate of B-16 melanoma solid cancer to BDF₁ mice inan amount of 0.25 ml per mouse. From the following day of thetransplantation, gnidimacrin and pimelea factor P₂ were administeredseparately once a day for 5 days. Each test group was compared inapothanasia rate with a control group. The results on gnidimacrin andpimelea factor P₂ are shown in Table 3 and Table 4, respectively.

                  TABLE 3                                                         ______________________________________                                                           Dosage   Apothanasia                                       Kind of cancer     (mg/kg)  rate (%)                                          ______________________________________                                        Lewis pulmonary cancer                                                                           0.02     40.0                                              B-16 melanoma      0.02     49.2                                              Colon 26 large bowel cancer                                                                      0.01     26.8                                              ______________________________________                                    

                  TABLE 4                                                         ______________________________________                                                           Dosage   Apothanasia                                       Kind of cancer     (mg/kg)  rate (%)                                          ______________________________________                                        Lewis pulmonary cancer                                                                           0.1      35.2                                              B-16 melanoma      0.1      41.8                                              Colon 26 large bowel cancer                                                                      0.05     18.5                                              ______________________________________                                    

In addition, gnidimacrin showed an apothanasia rate of 47.8% whenadministered intravenously to mice which had been transplanted WithLewis pulmonary cancer cells in an amount of 0.06 mg/Kg.

Test 4

Cells of gastric cancer, pulmonary cancer, liver cancer and leukemia,which had been separated from human patients, were separately placed onportions of RMP-1640 medium containing 10% calf serum and then incubatedat 37° C. for 4 days in a 5% CO₂ -incubator to obtain a cell populationof 10⁴ /ml. To the cells of each cancer kind so cultured, gnidimacrin orpimelea factor was added at varied concentrations, and the concentration(IC₅₀) of the test compound at which proliferation of the human cancercells was inhibited by 50% was determined. The results are shown inTable 5.

                  TABLE 5                                                         ______________________________________                                                        IC.sub.50 (μg/ml)                                          Site of cancer                                                                          Kind of cancer                                                                            Gnidimacrin                                                                              Pimelea factor                               ______________________________________                                        Gastric   MKU-28      0.006      0.01                                         cancer    MKV-74      >1         10                                                     K-III       0.0006     0.003                                                  MKV-45      0.0001     0.005                                        Pulmonary LU-99       1          10                                           cancer    N-237       0.2        3.8                                                    H-69        0.6        7                                                      PC-14       0.85       3.2                                                    PC-7        0.5        1.7                                          Liver cancer                                                                            HLE         2          10                                           Leukemia  K-502       0.0025     0.3                                          ______________________________________                                    

Test 5

Using laboratory animals, effects of gnidimacrin on solid cancer werestudied. First, intradermally inoculated to male BALB/c mice (each groupconsisting of 6 mice, each having a weight of 17-21 g) was 0.01 ml ofRPMI 1640 solution which contained 10% fetal serum and 1×10⁵ cells/ml ofMeth A fibrosarcoma. Seven days after the inoculation, solid cancer wasformed. Into the solid cancer, gnidimacrin which had been dissolved inphysiological saline containing 0.5% CMC, was administered at 0.001mg/Kg by injection. That administration was conducted once a day for 5days. On the 21st day from the inoculation of Meth A fibrosarcoma, thecancer was cut out and its weight was compared with that of a controlgroup not administered with gnidimacrin. As a result, it was found thatthe average cancer weight was 0.4 g for the gnidimacrin-administeredgroup but 2.1 g for the control group. Namely, gnidimacrin inhibited theproliferation of the solid cancer by 81%.

Preparation Example 1

    ______________________________________                                        Tablets                                                                       (Formulation)                                                                 ______________________________________                                        (1)    Corn Starch           48     g                                         (2)    Crystalline cellulose 42     g                                         (3)    Calcium carboxymethylcellulose                                                                      8      g                                         (4)    Light silicic acid anhydride                                                                        0.5    g                                         (5)    Magnesium stearate    0.5    g                                         (6)    Stelleramacrin A      1      g                                                Total                 100    g                                         ______________________________________                                    

(Procedures)

The above ingredients (1)-(6) were mixed uniformly according to theabove formulation and then compressed into tablets, of 200 mg each, by atableting machine.

Each tablet so obtained contains 2 mg of stelleramacrin A obtained inExample 1. Per adult, 1-10 tablets are administered a day in severalportions.

Preparation Example 2

    ______________________________________                                        Granules                                                                      (Formulation)                                                                 ______________________________________                                        (1)    Corn starch           88     g                                         (2)    Magnesium stearate    1.5    g                                         (3)    Calcium carboxymethylcellulose                                                                      8      g                                         (5)    Light silicic acid anhydride                                                                        1.5    g                                         (6)    Stelleramacrin B      1      g                                                Total                 100    g                                         ______________________________________                                    

(Procedures)

The above ingredients (1)-(5) were mixed uniformly according to theabove formulation and then compression-molded by a compression moldingmachine, followed by the crushing by a pulverizer and shifting intogranules.

The resultant granules contain stelleramacrin B, which had been obtainedin Example 2, in an amount of 10 mg/g. Per adult, 0.1-2 g of thegranules are administered a day in several portions.

Preparation Example 3

    ______________________________________                                        Capsules:                                                                     (Formulation)                                                                 ______________________________________                                        (1)     Corn starch         98.5   g                                          (2)     Light silicic acid anhydride                                                                      1.0    g                                          (3)     Stelleramacrin A    0.5    g                                                  Total               100    g                                          ______________________________________                                    

(Procedures)

The above ingredients (1)-(3) were mixed uniformly according to theabove formulation and No. 2 capsules were each filled with 200 mg of theresulting mixture.

Each capsule contains 1 mg of stelleramacrin A, which had been obtainedin Example 1. Per adult, 1 to 20 capsules are administered a day inseveral portions.

Preparation Example 4

    ______________________________________                                        Injection:                                                                    (Composition)                                                                 ______________________________________                                        (1)    Distilled water for injection                                                                       89.5   g                                         (2)    Soybean oil           5      g                                         (3)    Soybean phosphatide   2.5    g                                         (4)    Glycerin              2      g                                         (5)    Stelleramacrin A      1      g                                                Total                 100    g                                         ______________________________________                                    

(Procedures)

The ingredient (5) was dissolved in a mixture of the ingredients (2) and(3). To the resulting solution, the ingredients (1) and (4) were added,followed by emulsification, whereby an injection was obtained.

Industrial Applicability

As has been described above, stelleramacrin according to the presentinvention has high carcinostatic activities and can hence be employed asan anticancer agent applicable to a wide variety of cancer.

In addition, gnidimacrin- or pimelea-factor-P₂ -containing therapeuticsfor solid cancer are useful for the treatment of solid cancer such asgastric cancer, pulmonary cancer, liver cancer, mammary cancer and thelike.

We claim:
 1. Purified stelleramacrin A or purified stelleramacrin Brepresented respectively by the following formulas (I) and (II):##STR3##
 2. A process for the preparation of stelleramacrin A orstelleramacrin B, which comprises extracting a subterranean or aerialpart of Rui-xiang-lang-du with a lower alcohol, extracting the extractwith a water-insoluble organic solvent, isolating astelleramacrin-A-containing fraction or a stelleramacrin-B-containingfraction from the resulting extract, and then purifying the isolatedfraction.
 3. An anticancer agent comprising purified stelleramacrin A orpurified stelleramacrin B as an active ingredient.
 4. An anti-canceragent according to claim 3 which is effective for treating gastriccancer, pulmonary cancer, liver cancer or leukemia.
 5. A therapeuticagent for solid cancer, said therapeutic agent comprising an effectiveamount of purified gnidimacrin.
 6. A therapeutic agent for solid cancer,said therapeutic agent comprising an effective amount of purifiedpimelea factor P₂.
 7. A therapeutic agent according to claim 5, whereinsaid solid cancer is selected from gastric cancer, pulmonary cancer,liver cancer or fibrosarcoma.
 8. A therapeutic agent according to claim6, wherein said solid cancer is selected from gastric cancer, pulmonarycancer, liver cancer or fibrosarcoma.